Method to prepare sperm

ABSTRACT

Methods for improving the functionality and/or fertility of sperm, for example, by enhancing motility and/or extending the lifespan of sperm by subjecting the isolated sperm to a starvation protocol and/or ionophore are provided. Such methods may be used in, for example, artificial insemination to reduce the number of sperm needed for insemination and to improve conception rates.

PRIORITY

This application is a U.S. National Stage Filing under 35 U.S.C. 371from International Application No. PCT/US2017/025583, filed on Mar. 31,2017, and published as WO 2017/173391, which claims the benefit ofpriority from U.S. Provisional Patent Application Ser. No. 62/316,990,filed on Apr. 1, 2016, which are herein incorporated in their entiretyby reference.

GOVERNMENT GRANT SUPPORT

This invention was made with government support under HD038082 andHD044044 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Assisted reproductive technology (ART) includes such techniques as invitro fertilization (IVF), artificial insemination (AI),intracytoplasmic sperm injection (ICSI) (other techniques usingenucleated cells) multiple ovulation and embryo transfer (MOET) and ART(as well as other embryo transfer techniques), is used across the animalkingdom, including humans and other animals. ART methods are usuallyexpensive, time consuming and marginally successful given the inherentfragility of gametes and embryos when outside of their naturalenvironments. Furthermore, the use of ART within the animal breedingindustry in a commercially feasible manner is additionally challengingdue to the limited availability of genetically desirable gametes andzygotes. One way to lower the cost of ART and to improve its commercialfeasibility is to increase the efficiency of the involved processes byimproving the viability and overall quality of gametes, zygotes andembryos.

For example, in conventional AI, one problem limiting its commercialapplication in certain species is the need to use extremely high numberof sperm cells per AI dose to ensure successful fertilization.Similarly, in IVF, the percentage of zygotes that develop into embryosremains frustratingly low; this high rate of loss significantlyincreases the cost of embryos and related services to end-users.

SUMMARY OF THE INVENTION

The invention is directed to a novel method of treating sperm forartificial reproductive techniques including in vitro fertilization,ICSI, and artificial insemination such as intrauterine insemination(IUI) and intravaginal insemination (IVI). Each species can benefit fromthis technology, for example, improvement of IVF, ICSI and artificialinsemination for humans; IVF for horses; maintenance of live sperm inextenders for pigs; improvement of ART for mice genetic models; and forall species, improvement of embryonic development after fertilization.For example, benefits include significantly improved percentage ofsuccess fertilization and/or embryonic development in all species. Or,for example, such as horse IVF, the method is unique as IVF in thisspecies has not been achieved.

The present invention is based on the surprising finding that reducingintracellular energy molecules including, but not limited to ATP, usinga nutrient starvation protocol carried out on isolated sperm canincrease sperm functionality and fertility rates, as well as embryodevelopment to blastocysts rates and that those blastocysts whentransferred to a female increased pregnancy rates. Also, we have thesurprising finding that treatment with calcium ionophore, such asA23187, for a short time period, in addition to increasing spermmotility and fertilization rates, A23187 significantly increased embryodevelopment rates to blastocysts (Scientific Reports 6, Article number:33589 (2016)). Accordingly, one embodiment of the present inventioncomprises a method of treating sperm cells by exposing sperm cells toconditions of temporary starvation obtained by removing energysubstrates (which include, but are not limited to, glycolytic substratesand Krebs cycle substrates such as glucose, fructose, pyruvate, lactate,citrate or a combination thereof from sperm surrounding media), exposingthe sperm cells to an ionophore and/or combining these procedures indifferent (any) order.

One embodiment provides a method to increase sperm functionalitycomprising a) isolating sperm; b) removing, or not, some or allendogenous energy nutrients including, but not limited to, glycolyticsubstrates and Krebs cycle substrates such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof; c) placing saidsperm in a media with reduced or no added energy nutrients (as definedin b)) for a period of time dependent on the species underconsideration; and d) after (b and c), adding an energy nutrient (whichis any energy substrate including but not limited to glycolyticsubstrates and Krebs cycle substrates such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof) to said media andsperm, so as to increase sperm functionality as compared to sperm cellsthat had not undergone energy nutrient starvation.

One embodiment provides a method to increase Artificial Inseminationpregnancy rates comprising a) isolating sperm; b) removing, or not, someor all endogenous energy nutrients including but not limited toglycolytic substrates and Krebs cycle substrates such as glucose,fructose, pyruvate, lactate, citrate or a combination thereof; c)placing said sperm in a media without an energy nutrient (as defined inb)) for a period of time; d optionally adding an energy nutrient (whichis any energy substrate including but not limited to glycolyticsubstrates and Krebs cycle substrates such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof) to said media andsperm of b) and/or c); and e) using said sperm from b), c) and/or d) forintrauterine (IUI) or vaginal insemination (IVI).

Another embodiment provides a method to increase fertility in vitrocomprising a) isolating sperm; b) removing, or not, some or allendogenous energy nutrients including, but not limited to, glycolyticsubstrates and Krebs cycle substrates such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof; c) placing saidsperm in a media without an energy nutrient (which is any energysubstrate including, but not limited to, glycolytic substrates and Krebscycle substrates such as glucose, fructose, pyruvate, lactate, citrateor a combination thereof) (for a period of time dependent on the speciesunder consideration); d) adding an energy nutrient (as defined in b andc) to said media and sperm of b and/or c); and e) contacting said spermwith an ovum of the same species as the sperm, so as to increasefertility as compared to a method where sperm cells have not undergoneenergy nutrient starvation.

Another embodiment provides a method to increase fertility usingintracellular sperm injection (ICSI) comprising a) isolating sperm; b)removing, or not, some or all endogenous energy nutrients including, butnot limited to, glycolytic substrates and Krebs cycle substrates such asglucose, fructose, pyruvate, lactate, citrate or a combination thereof;c) placing said sperm in a media without an energy nutrient (which isany energy substrate including, but not limited to, glycolyticsubstrates and Krebs cycle substrates, such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof) (for a period oftime dependent on the species under consideration); d) optionally addingan energy nutrient (defined in b and c) to said media and sperm of b);and e) injecting the sperm of b), c) or d) inside an ovum of the samespecies as the sperm, so as to increase fertility as compared to amethod where sperm cells have not undergone energy nutrient starvation.

Another embodiment provides a method to increase embryo qualitycomprising a) isolating sperm; b) removing, or not, some or allendogenous energy nutrients including, but not limited to, glycolyticsubstrates and Krebs cycle substrates such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof; c) placing saidsperm in a media without an energy nutrient (which is any energysubstrate including, but not limited to, glycolytic substrates and Krebscycle substrates such as glucose, fructose, pyruvate, lactate, citrateor a combination thereof); d) adding an energy nutrient (defined as inb) and c)) to said media and sperm; e) contacting said sperm with anovum of the same species as the sperm; and f) allowing said sperm andovum to develop into a blastocyst, so as to increase embryo quality ascompared to a method where sperm cells have not undergone energynutrient starvation.

Another embodiment provides a method to increase embryo qualitycomprising a) isolating sperm; b) removing, or not, some or allendogenous energy nutrients including, but not limited to, glycolyticsubstrates and Krebs cycle substrates such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof; c) placing saidsperm in a media without an energy nutrient (e.g., metabolic nutrient asdefined in b)); d) optionally adding an energy nutrient (as defined inb)) to said media and sperm of b); e) injecting the sperm of b), c) ord) inside an ovum of the same species as the sperm; and e) allowing saidsperm and ovum to develop into a blastocyst, so as to increase embryoquality as compared to a method where sperm cells have not undergoneenergy nutrient starvation.

In one embodiment, removal of energy nutrients from biological fluidswill be done by washing the sperm using centrifugation techniques withmedia lacking metabolic nutrients (including, but not limited to,glycolytic substrates and Krebs cycle substrates such as glucose,fructose, pyruvate, lactate, citrate or a combination thereof).Depending on the species, the centrifugation procedure includes one, twoor more washes.

In one embodiment, removal of energy nutrients from biological fluidsincluding epididymal and seminal fluid will be done by passing the spermthrough materials such as gel filtration resins (e.g. Sephadex®) orion-exchange resins (e.g. DOWEX, DEAE). These resins will be used withthe goal of removing metabolic nutrients including, but not limited to,glycolytic substrates and Krebs cycle substrates such as glucose,fructose, pyruvate, lactate, citrate or a combination thereof from thesaid biological fluids.

In one embodiment, removal of energy nutrients will be done usingdensity gradients lacking energy nutrients including but not limited toPercoll® gradients.

In one embodiment, the sperm are in an energy nutrient (as definedherein) absent environment (step b and/or c) for any period of time(such as from about 1 minute to several hours, including about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours,about 3 hours, about 4 hours, about 5 hours and so on, including about18-24 hours).

In one embodiment, the energy nutrient added to said media in step d) isany energy substrate including, but not limited to, glycolyticsubstrates and Krebs cycle substrates such as glucose, fructose,pyruvate, lactate, citrate or a combination thereof.

In one embodiment, no energy nutrient will be added back in d). Spermwill be used for any assisted reproductive technique while in starvingmedia lacking metabolic nutrients.

In one embodiment, decrease of intracellular energy pools in the form ofATP or other energy molecules will be obtained using inhibitors of anyof the enzymes of glycolysis, Krebs cycle or mitochondria oxidativephosphorylation. In this embodiment, said reagents can be used alone orin combination with the starving protocols described above.

In one embodiment, decrease of intracellular energy pools (e.g. ATP)will be induced by incubation of sperm in the absence of divalentcations including, but not limited, to calcium and magnesium. In theabsence of these cations, there is an influx of sodium ions towards theintracellular sperm compartments. To eliminate this excess of sodium,the sperm use high levels of ATP and reduce the total amount of ATP.Elimination of divalent cations can be done by eliminating them frombiological fluids such as seminal fluid, by not adding the divalentcations to the incubation media, and/or by adding divalent cationchelators including, but not limited to, EDTA and EGTA. Elimination ofdivalent cations for assisted reproductive techniques including, but notlimited to, IVF, ICSI, IUI and IVI, can be done alone or in combinationwith the starving protocol.

In one embodiment, the sperm is vertebrate, including mammalian,including, but not limited to human, murine, avian (poultry), bovine,porcine, ovine, camelids (e.g. alpaca) or equine.

In one embodiment, the sperm cells are exposed to an ionophore, such asa calcium ionophore. This embodiment will be used alone or incombination to starving protocols.

One embodiment provides the use of sperm, prepared according to themethods described herein, with the purpose of producing geneticallymodified species (including, but not limited, to mouse) using techniquessuch as gene editing (e.g. TALEN, CRISPR/CAS) or any other transgenic,knock-out/in technology in eggs, zygotes and other embryonic stages,including early embryonic stages such as morula and blastocyst as wellas post-implantation.

One embodiment provides the use of sperm, prepared according to themethods described herein, as a vector to introduce DNA and/or RNAmaterial in the egg by artificial insemination, in vitro fertilizationor ICSI, with the purpose of producing genetically modified species (insome embodiments with the aid of techniques such as gene editing (e.g.TALEN, CRISPR/CAS) or any other transgenic, knock-out/in technology ineggs, zygotes and other embryonic stages, including early embryonicstages such as morula and blastocyst as well as post-implantation).

Thus, the invention provides a method for improving the functionalityand/or fertilizing capability of sperm cells by subjecting them toreduced levels of intracellular energy in the form of ATP or otherenergy substrates. This decrease in ATP will be produced by a period ofstarvation, use of inhibitors of glycolytic, Krebs cycle, or oxidativephosphorylation, by incubation of sperm in media without divalentcations (achieved by elimination of divalent cations from incubationmedia, by addition of divalent cation chelators (including EDTA orEGTA), or by combination of these procedures), or by a combination ofthe said methodologies. The invention further comprises treating spermcells with or without an ionophore, such as a calcium ionophore,optionally in combination with any of the methods described herein withthe purpose of improving embryo development and pregnancy rates.

One embodiment provides a new Sperm Conditioning Medical Device, whichcan be assembled as a commercially available kit to improve AssistedReproductive Technology (ART). The general translational objective ofthe invention is to generate a new ART technology to be applied in IVF,ICSI and AI in humans, as well as in the biomedical research industry ofanimal models for human diseases, and in the breeding industry. Inparticular, disclosed herein are sperm media conditions, particularlyfor the use in human sperm, as well as a sperm conditioning device thatwill allow for sperm treatment, and for changes in the sperm-containingsuspension without the use of centrifugation. This new method/device hasthe potential of replacing current standard media and of revolutionizingART practices worldwide. Specifically, a sperm-compatible, plasticcolumn of approximately 2-5″×0.5″ (L×W), and 10-ml total capacity ispackaged with a gel filtration slurry such as Sephadex® G-15 or SephadexG-25 which will allow for separation of the sperm cell fraction (largersize) from the low molecular weight components present in seminal fluid(or a sperm sample from other sources). The base of the column can beprovided with a porous lining of either glass wool or a filteringmembrane; this will be optional and/or depending on sperm species. As analternative, a dialysis-based device from proper material and ofappropriate pore size can be used. As another alternative, ion-exchangeresins including, but not limited to, DOWEX, can be used instead of gelfiltration. These known sperm medium components, of a much smaller MW,play a role metabolically in sperm motility and fertilizing capacity. Ina first step, the sperm sample will be passed through the device, inwhich the slurry of Sephadex® G-15 or Sephadex G-25 is free of thosecomponents, labeled as Solution A. After a 45-60 min incubation, thesperm will be recovered in Solution B, which does contain thosemetabolically components. This metabolic switch allows for a highlycompetent sperm sample, with an increased motility and fertilizingcapacity, and significantly improved pregnancy rates and potential forhealthier embryo development.

In one embodiment, a kit is adapted to the needs of each species. Suchkits can include generation of kits for better sperm conservation inextenders; kits for artificial insemination in all animal speciesincluding humans; kits for in vitro fertilization; kits for ICSI; andkits for treating sperm produced in vitro from stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a motility assay.

FIG. 2 depicts a prepared tissue culture dish for the IVF experiment.

FIG. 3 depict images of embryos from two cells to blastocyst stage FIG.4 depicts a mouse proestrus and with vaginal plug.

FIGS. 5A-5F show that A23187 improves hyperactivation and fertilizingcapacity of sperm from B57BL6 (black 6) genetic background. Mouse spermwere incubated in Hepes-TYH (western blotting) or TYH standard (Motilityand IVF assay) and KSOM for embryo culture. (A) CD-1 and C57BL6 micesperm hyperactivation in 60 min with or without A23187 pre-treatment.(B) CD-1 and C57BL6 mice in-vitro fertilization rate with or withoutA23187 pre-treatment after 4 hours of insemination. (C) Developmentalstage from eggs fertilized by C57BL6 sperm with A23187 pre-treatment.(D) A23187 pre-treatment over comes the fertilization inhibition byH-89. (E) PKA activation in spermatozoa with a concentration of A23187(20 uM) pre-treatment. (F) The addition of H-89 (50 uM) inhibited PKAactivation in spermatozoa with or without A23187 pre-treatment.

FIGS. 6A-6D demonstrate that A23187 treatment induced hyperactivationand fertilizing capacity of CatSper1 KO sperm. Mouse sperm wereincubated in Hepes-TYH (western blotting) or TYH standard (Motility andIVF assay) and KSOM for embryo culture. (A) Catsper KO mouse spermhyperactivate in 60 minutes with A23187 pre-treatment. (B) Catsper WTand KO mice in-vitro fertilization rate with or without A23187pre-treatment. (C) Developmental stage from eggs fertilized by CatsperKO sperm rate with A23187 pre-treatment and pups obtained from CatsperKO sperm treated with A23187. (E) Genotyping of F2 Pups from Catsperheterozygous obtained from Catsper K.O rescued with A23187.

FIGS. 7A-7E show that A23187 treatment also induced fertilizing capacityin sperm from sAC and SLO3 sterile KO genetic models, but not in spermfrom PMCA4 KO. Mouse sperm were incubated in Hepes-TYH (westernblotting) or TYH standard (Motility and IVF assay) and KSOM for embryoculture. (A) Sperm from C57BL6, SLO3 KO, and SAC 1-2 KO were pre-treatedwith or without A23187 and the percentage of motility was obtained after60 min of capacitation. (B) C57BL6, SLO3 KO, and SAC 1-2 KO spermincrease hyperactivation after 60 min upon A23187 pre-treatment. (C)Also SLO3 KO, and SAC 1-2 KO fertility rates are rescued when sperm arepre-treated with A23187. (D-E) Plasma membrane Calcium ATPase pump 4efflux pump KO (PMC4) was used as a control. A23187 could not rescuehyperactivation and fertility rates.

FIGS. 8A-8D depict starving conditions induced loss of phosphorylationpathways and motility. After incubation in the absence of nutrients,addition of nutrients rescued all parameters and improved motility andhyperactivation over controls. In these experiments, sperm were obtainedfrom C57B16/j male mice. A and B. Measurement of PKA activation usinganti phosphoPKA substrate antibodies (A) and the increase in tyrosinephosphorylation (B). Sperm were incubated in the absence of HCO3− andBSA (non capacitating conditions), or in the presence of these compounds(capacitating conditions) for 1 hour and in the presence or in theabsence of glucose and pyruvate as indicated. After 1 hour, aliquots ofsperm incubated in the absence of glucose and pyruvate (starvingconditions), were supplemented with glucose (5 mM), pyruvate (0.5 mM) orboth. C and D. Aliquots of sperm treated using the same protocol asdescribed in A and B were evaluated for motility (C) and hyperactivatedmotility (D) using CASA.

FIGS. 9A-9F depict starving plus rescue sperm incubation increasedfertilization rates and embryo development rates in mouse sperm. Spermobtained from C57BL6 mouse strain from different age mice (as shown infigure) were incubated in capacitating TYH media in the presence(control) or in the absence of glucose and pyruvate (starving+rescue).After 40 min, sperm in starving conditions are rescued by addition ofglucose (5 mM) and pyruvate (0.5 mM). Sperm in both conditions are leftfor additional 20 min and then added to the insemination dropletcontaining cumulus enclosed CD1 oocytes (A, B and C) or C57BL6 (D, E andF). Number of repetitions (independent mice) is given below eachtreatment. Percentage of fertilization considers the number of oocytesthat achieved 2-cell stage (A and D). 2-cell embryos are thentransferred to KSOM media and further incubated for additional days.Percentage of blastocyst is calculated either by considering the numberof 2-cell embryos (B and E) or by considering the initial number ofoocytes (C and F).

FIGS. 10A-10D depict starving plus rescue method improves blastocystcell number, outgrowth and number of pups per embryo transferred. A.Blastocyst cell number. Sperm were incubated in control or starved plusrescue (S+R) conditions and used for in vitro fertilization. Two-cellembryos were then transferred to KSOM media and further incubated for atotal of 3.5 days. Blastocysts were then stained with Hoecsht and thenumber of cells in each blastocyst counted. Numbers represent theaverage±SEM (n=10). B. Blastocyst in vitro outgrowth. Blastocystsobtained with control or starved plus rescue sperm were assayed foroutgrowth in vitro (n=10). C. Litter size obtained with the differenttreatments analyzed by age group. Blastocysts obtained from spermincubated in control or starved plus recue conditions were transferredto pseudo-pregnant females. The analysis was done separating the resultsinto two groups (sperm form mice 2-12 month old (n=15) and sperm frommice 12-24 month old (n=8). Each data point is presented in the graph.D. Percentage of pups per number of embryos transferred. The same datawere analyzed considering the number of pups that were born consideringthe respective number of blastocysts transferred in each case. Each datapoint is presented in the graph.

FIGS. 11A-11C. IUI is improved using starved sperm. Sperm from C57BL6mice were incubated in control media or in starving media (starved).Once sperm are not moving (about 40 min), sperm are transferrednon-surgically to pseudo-pregnant females. A. percentage of females thatbecome pregnant after IUI with sperm incubated in either control orstarved media. B. Average litter size±SEM (n=10). C. Example of pupsobtained by IUI using starved method.

FIGS. 12A-12C. Starved plus rescue treatment improves fertilizationrates and embryo development from sub-fertile strains. A. Fertilizationrate of FerTDR/DR sperm incubated under control, transient exposure toA23187 CA2+ ionophore, or starved plus rescued protocols. Datarepresents average±SEM (n=6). B. Embryo development rates. Percentage ofblastocysts obtained from two-cell embryos under the same conditionsdescribed in A. C. Fertilization and embryo development rates of Akitaand SJL/J mice strains. Sperm were treated in control or starved plusrescued condition. The table indicates the number of oocytes used in 4independent experiments, together with the number of cells that reachtwo-cell stage with the respective percentage. Two-cell embryos weretransferred to KSOM media and further incubated for 3.5 days. The numberof blastocysts obtained with the respective percentage of blastocystsfrom two-cell embryo is given. Finally, the last column represents theeffectiveness of each treatment given by the percentage of blastocystsfrom the initial number of oocytes used in the assays.

FIGS. 13A-13B. Combination of starved plus rescued protocols with thetransient exposure to CA2+ ionophore A23187 rescued the completelysterile phenotype of CatSper KO mice. A. Fertilization Rate. Sperm fromCatSper1 KO mice were incubated in four different conditions: 1)control; 2) A23187 transient treatment; 3) starved plus recue treatment;and 4) starved plus rescue treatment followed by A23187 transienttreatment. B. Blastocyst development. Two-cell embryos obtained in Awere transferred to KSOM media and further incubated for 3.5 days. Thepercentage of blastocyst with respect to the two-cell embryos arepresented. In both A and B, the results presents the average±SEM form 4independent CatSper KO mice (n=4).

FIGS. 14A-14D. Bovine IVF is improved when sperm are treated withmetabolically-enhanced media. Frozen bovine sperm were thawed andincubated in control IVF media or in metabolically-enhanced IVF media(MEM). In vitro fertilization was conducted with eggs from ovariesobtained from slaughter houses and matured in vitro. Notice thatdifferent to mouse eggs, the quality of these eggs is not homogeneousand may influence in vitro fertilization from the egg side. A. IVF wasassessed by counting the percentage of oocytes that reach the two-cellembryo stage. B. Development was assessed by evaluating the percentageof 2-cell embryos that reach blastocyst stage. C. 2 blastocysts wereobtained with control IVF. D. 4 blastocysts were obtained usingMEM-treated sperm.

FIGS. 15A-15B. Calcium oscillations elicited by intracellular-sperminjection (ICSI) are enhanced when sperm are treated withmetabolically-enhanced media. Frozen bovine sperm were thawed andincubated in control IVF media or in metabolically-enhanced IVF media(starved plus rescue). ICSI was conducted using eggs from ovariesobtained from slaughter houses and matured in vitro. Oocytes werepreviously loaded with the calcium dye Fura 2. Oscillations weremeasured for six hours after sperm injection. A. Calcium oscillationsafter injections of starved plus rescue-treated bovine sperm. B. Calciumoscillations after injection of control bovine sperm.

FIG. 16. Starved and rescue protocol improves two-cell and blastocystdevelopment when bovine sperm are used in ICSI. Frozen bovine sperm werethawed and incubated in control IVF media or following the starved andrescue protocol. ICSI was conducted using eggs from ovaries obtainedfrom slaughter houses and matured in vitro. A. IVF was assessed bycounting the percentage of oocytes that reach two-cell embryo stages. B.Development was assessed by evaluating the percentage of 2-cell embryosthat reach blastocyst stage.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention. Specific and preferred values listedbelow for radicals, substituents, and ranges are for illustration only;they do not exclude other defined values or other values within definedranges for the radicals and substituents.

As used herein, the articles “a” and “an” refer to one or to more thanone, i.e., to at least one, of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

The term “isolated” refers to a factor(s), cell or cells which are notassociated with one or more factors, cells or one or more cellularcomponents that are associated with the factor(s), cell or cells invivo.

In relation to sperm, it should be understood that the terms “activity”and/or “function” encompass physiological processes such as, forexample, sperm motility, sperm tropism (namely, the tendency of sperm tomove towards or away from certain stimuli), capacitation (understood asthe gaining of the ability to fertilize) and fertilizing ability. Theterms “activity” and/or “function” may further include processes whichoccur prior to and during fertilization and/or interaction with the egg(or membranes/layers thereof)—such processes may include, for examplesperm capacitation and acrosomal activity.

With regard to sperm motility, one of skill will appreciate that theterm “motility” not only relates to general movement, but may be appliedto other aspects of motility such as, for example, the speed of movementof a sperm cell and/or any increase or decrease in the proportion ofmoving sperm cells in any given population. It also applies to aspecialized type of motility known as “Hyperactive motility orhyperactivation” which encompass changes in the symmetry of the spermflagellum movement as well as in the force generated by such movement.As such, the PDEIs described herein may be used not only to increasesperm motility, but also to increase the speed of movement of a spermcell, the changes in symmetry of the flagella, the changes in the forcegenerated by movement and/or the proportion of moving and hyperactivecells in any given population of sperm.

The terms “comprises,” “comprising,” and the like can have the meaningascribed to them in U.S. Patent Law and can mean “includes,” “including”and the like. As used herein, “including” or “includes” or the likemeans including, without limitation.

Sperm

Sperm cell quality may refer to any one or a combination of the variousattributes of sperm cells previously mentioned or further mentionedherein, such as, for example, viability, motility, functionality,stimulation, and preservation of the sperm, or fertility rates,insemination rates, or fertilization rates corresponding to the sperm(such as in the fertility of the sperm). Sperm cell characteristic mayrefer to any one or a combination of various biological, chemical,physical, physiological, or functional attributes of one or more spermcells, such as chromosome bearing attributes of the cell, or in someembodiments may refer to sperm cell quality as previously described.

Sperm Sample Collection

The sperm sample may be a freshly collected sample from a source animal,such as bovine, equine, porcine, murine, human, or other vertebratesource including mammals, or a thawed, previously cryopreserved sample.Moreover, the sample may be a single ejaculate, multiple pooledejaculates from the same mammal, or multiple pooled ejaculates from twoor more animals. It can also be directly collected from any section ofthe male reproductive tract including testicular sperm, and spermobtained from caput, corpus or cauda epididymis.

Various collection methods are known and include the gloved-hand method,use of an artificial vagina, and electro-ejaculation. The sperm arepreferably collected or quickly transferred into an insulated containerto avoid a rapid temperature change from physiological temperatures(typically about 35° C. to about 39° C.). The ejaculate typicallycontains about 0.5 to 15 billion sperm per milliliter, depending uponthe species and particular animal. However, the number of sperm could bereduced because of subfertile or infertile phenotypes. In some cases,the sperm are directly taken from testicular or epididymal tissue usingdifferent methodologies such as puncture of the testis or epididymisusing surgical procedures or removing the testis or epididymis andcollecting the sperm in surrounding media.

Regardless of the method of collection, an aliquot may be drawn from thesperm sample and evaluated for various characteristics, such as forexample, sperm concentration, sperm motility, sperm progressivemotility, sample pH, sperm membrane integrity, and sperm morphology.This data may be obtained by examination of the sperm using, forexample, the Hamilton-Thorn Motility Analyzer (IVOS), according tostandard and well known procedures (see, for example, Farrell et al.Theriogenology (1998) 49(4): 871-9; and U.S. Pat. Nos. 4,896,966 and4,896,967).

Dilution/Media

The sperm sample may be combined with a buffer (in the form of a solidor solution) to form a sperm suspension. Among other things, the buffermay enhance sperm viability by buffering the suspension againstsignificant changes in pH or osmotic pressure. Generally, a buffer isnon-toxic to the cells and is compatible with the dye used to stain thecells. Exemplary buffers include phosphates, diphosphates, citrates,acetates, lactates, and combinations thereof. Examples of such buffersinclude TRIS, TCA, TEST, bicarbonate/CO₂, sodium citrate, HEPES, TL,TES, citric acid monohydrate, HEPEST (Gradipore, St. Louis, Mo.), PBS(Johnson et al., Gamete Research, 17:203-212 (1987)), and Dulbecco's PBS(Invitrogen Corp., Carlsbad, Calif.).

One or more buffers may be combined together or with additives to form abuffered solution, and the buffered solution combined with the spermsample to form a sperm suspension.

In addition to a buffer, the sperm suspension may also contain a rangeof additives to enhance sperm viability or motility. Exemplary additivesinclude energy sources, protein sources, antibiotics, and compositionswhich regulate oxidation/reduction reactions intracellularly and/orextracellularly. One or more of these additives may be introduced intothe buffer or buffered solution before the formation of the spermsuspension or, alternatively, may be separately introduced into thesperm suspension.

To minimize dilution shock, provide support to the cells, or dispersethe cells throughout the suspension, a protein source may also beincluded in the buffer, buffered solution, or sperm suspension.Exemplary protein sources include egg yolk, egg yolk extract, milk(including heat homogenized and skim), milk extract, soy protein, soyprotein extract, serum albumin, bovine serum albumin, human serumsubstitute supplement, and combinations thereof.

An antibiotic may be added to the sperm suspension in order to inhibitbacterial growth. Exemplary antibiotics include, for example, tylosin,gentamicin, lincomycin, spectinomycin, Linco-Spectin® (lincomycinhydrochloride-spectinomycin), penicillin, streptomycin, ticarcillin, orany combination thereof. The Certified Semen Services (CSS) and NationalAssociation of Animal Breeders (NAAB) have promulgated guidelinesregarding the use of antibiotics with respect to sperm collection anduse.

A composition which regulates oxidation/reduction reactionsintracellularly and/or extracellularly may also be included in the spermsuspension. Such a composition may provide a protective effect to thesperm cells, such as for example by maintaining sperm viability orprogressive motility. Examples of such a composition include, forexample, pyruvate, vitamin K, lipoic acid, glutathione, flavins,quinones, superoxide dismutase (SOD), and SOD mimics. If included in thesperm suspension, such a composition may be present in a concentrationsufficient to affect the protective effect without detrimentallyaffecting sperm health.

Nutrient Starvation Method

In the method disclosed herein, isolated sperm cells are placed inconditions absent energetic nutrient compounds. For example, most mediathat sperm cells are placed in contain glucose, lactate and/or pyruvate,which are energetic compounds. If such compounds are removed, the spermcells are essentially starved because they lack energy sources. Wheneach one is added back in singly, their individual role can bedetermined. It was determined that the sperm cells were not dead afterbeing placed in a media free of energetic compounds. Rather, they juststopped swimming and appeared completely immotile. It was determinedthat glucose is more important than pyruvate as an energy source formouse sperm. However, in other species, such as bovine, mitochondrialKrebs cycle and oxidative phosphorylation are more relevant.

Nutrient(−) Nutrient (+) removing any type of adding any type ofcarbohydrate/sugar/energy carbohydrate/sugar/energy nutrient yieldsnutrient increased motility after starvation/removal of sugar/energynutrient

Surprisingly, the sperm not only survive the starving process, but arevery active. Even more surprisingly, they actually increase inactivity—hyperactivated motility/hyperactivation. This is very good forfertilization. They also changed their motility pattern; in that theymove very fast and the movement is more asymmetric. This led toincreased IVF rates as compared to control (IVF without starvation ofsperm cells prior to IVF) when sperm from a suboptimal strain of mice(CBL57, black six) were used.

For example, CDI mice have a good fertilization rate to begin with,however, with the starvation method, the rate of zygotes going toblastocyst improved. In addition, the overall success of embryodevelopment already good in CD1 mice improved; thereby showing anincrease in embryo health. Although sperm of these mice are already goodfor IVF and embryo development, other mice strains have suboptimalfertilization and embryo development rates. Two cases assayed wereC57BL/6, black six and Balb6. These mice naturally show poor rate forreproduction in vivo and in vitro and only 35% arrive to blastocyst,with approximately a 50% fertilization rate. However, with starvationmethod, both strains of mice show 90% and up to 100% go to blastocyst.This is a vast improvement and very surprising. It is believed that asperm issue is the cause of balb6 and C57BL/6 mice not being goodreproducers. With the sperm starvation protocol described herein,fertilization and embryo formation are greatly improved.

In the starvation protocol, isolated sperm are placed in an energynutrient absent environment for a period (for example, until the spermloose progressive motility) that could last from the starting point ofthe incubation in starving media to several hours depending on thespecies, including immediate contact up-to many seconds, minutes, hoursor days. For example in mouse sperm the time to stop motility is between30 min and 1 hour. In bull and human ejaculated sperm is between 3 and 5hours. The time frame of incubation in starving media will depend on thespecies. The method can also be used to extend the life of sperm inextenders with limited amount of energy sources. In those cases, theembodiment contemplates suspending sperm treated or not with thestarving procedure in media that contain zero or low concentrations ofenergy substrates.

The energy nutrient can be any agent/molecule that can provide energy orbe used as energy by the sperm cells; this includes, but is not limitedto, carbohydrates or sugar, including monosaccharides (such as fructose,glucose, galactose and mannose) and disaccharides (sucrose, lactose,maltose, and trehalose), as well as polysaccharides, galactose,oligosaccharides, polymers of sugar, glucose, pyruvate and combinationsthereof. The energy nutrient can also be sodium lactate and lactic acid.Also, any other metabolizable molecule (e.g., any metabolite that hasthe potential to be converted in a source of energy including ATP, ADP,AMP, analogues of these compounds or compounds that could be convertedin ATP, ADP or AMP) such as lipids, amino acids, nucleotides, etc.

Assisted Reproductive Technology (ART)

ART is the technology used to achieve pregnancy in procedures such asfertility medication, artificial insemination, in vitro fertilizationand surrogacy. It is reproductive technology used primarily forinfertility treatments, and is also known as fertility treatment. Itmainly belongs to the field of reproductive endocrinology andinfertility, and may also include intracytoplasmic sperm injection(ICSI) and cryopreservation. Some forms of ART are also used with regardto fertile couples for genetic reasons (pre-implantation geneticdiagnosis).

The cost for fertility investigation and treatments can be great andmany times insurance does not cover such procedures.

A) Artificial Insemination, IVF and ICSI

Artificial insemination in mice carried out with the starvation protocoldescribed herein in which sperm were starved prior to use led to 55% offemale pregnant, whereas control AI without starvation, led to only 10%of pregnancy. Moreover, litter size from pregnant females using starvingsperm was on average 6 pups while pregnant females obtained with controlsperm only deliver an average of 2 pups. Therefore, the protocol notonly led to increased motility, but also increased fertilityrates/ability to fertilize. Thus, the use of the sperm starvationprotocol in humans can lead to the use of more artificial inseminationprocedures rather than IVF or ICSI.

IVF in humans is costly, easily about $15,000-$17,000 USD per try. InIVF, after fertilization, the cells are grown to the blastocyst stageand then implanted. Thus, not only fertilization and fertilization ratesare important, but also rates of cells that continue on to blastocystare important (improve embryo quality). The sperm cell starvationprotocol described herein leads to an increase in both.

For Intracellular sperm injection (ICSI), it does not matter if thesperm are not motile. Thus, one would believe that a starvation protocolwhich leads to increased motility would not be needed. Surprisingly, inaddition to fertility rates, embryo quality increased with thestarvation protocol after conducting ICSI in bovine eggs. Thisimprovement in bovine is very relevant because this species is known tobe resilient to ICSI treatment. Maximum blastocyst formation using ICSIin bovines has been reported by many laboratories to be not more than5%. Using the starving protocol, sperm injected using ICSI technologyachieved 50% of cleavage (two cells).

In conclusion, the sperm cell starvation protocol is a method thatimproves in vitro fertilization, embryo quality, and artificialinsemination.

B) Uses In Vitro in Infertility Clinics

Procedures used in infertility clinics to prepare human sperm samplesfor either in vitro fertilization, ICSI or intrauterine insemination caninvolve the starvation protocol described herein to prepare spermsamples prior to their use.

C) Agricultural Applications

The present invention is applicable to stimulating fertilizing abilityof sperm in domestic animals. In many agriculturally important species(e.g., cattle, pigs, sheep) artificial insemination using either freshor frozen/thawed semen samples is used to establish pregnancies. This isparticularly important in controlled breeding programs where it iscommercially advantageous for farmers to have specificgenetically-determined traits introduced into their stock. Use of themethods described herein will result in improved pregnancy rates.Mammalian sperm are frequently damaged by freezing and thawing andresults in lower fertility. By improving the performance of the viablesperm, the starvation protocol for sperm preparation used forinsemination may promote a higher pregnancy rate per estrus cycle,reducing the number of cycles required to ensure conception and hencereducing the overall cost of artificial insemination. At the same time,semen from animals with highly desirable traits could be used toinseminate more females because fewer cycles would be needed to ensureconception in any one female.

D) Exotic Animals.

In zoos all over the world, reproduction of exotic species in captivityor in the wild is a relevant goal. The methods described hereinincluding starving can be used to improve artificial insemination, IVFor ICSI in exotic species. In addition to those animals maintainedcaptive in a zoo, conservation programs aim to improve reproduction inanimals that are close to extinction in the wild. The methods describedherein can be used for this purpose.

The following examples are intended to further illustrate embodiments ofthe invention and are not intended to limit the scope of the inventionin any way.

EXAMPLES Example I: Starvation Protocol

Materials

Males CD1 male 3-8 months old (or retired breeder) or C57BL6 mice;Females CD1 or C57BL6 6-8 weeks old; Hormones PMSG (G4877) y hCG(C1063); Filter (Sterivex 0.2 μm Millipore); Syringe (10 ml to filtermedia and 1 ml to inject hormones); BSA (Sharlip et al.), TL-HepesMedium; TYH Standard; TYH Standard Free (Glucose and Pyruvate free); BSA(Sigma); 50 ml Falcon tubes; 15 ml Falcon tubes; 2 ml Falcon Tubes; 2 mldishes; Tissue Culture dish 35×10 mm (Falcon ref 353001); Glassmicrocapilar (pipette); Aspirator tube; light mineral oil Fetus BovineSerum (Atlanta Biologicals cat# S11150H); KSOM ((cat# MR-106-D))

Methods

Motility Assay (FIG. 1)

-   -   1. Sacrifice male mouse via dislocation or CO₂ chamber.    -   2. Open the abdomen with fine scissors. Begin from the pelvic        area and make a V shape to see all the organs    -   3. Look for the testis (white pale balls) and follow the        seminiferous tubules until you find the cauda of the epididymis        (looks like a small brain).    -   4. Take the cauda epididymis and make three or four incisions        until you see white fluid coming out.    -   5. Put cut 1 epididymis in 2 ml modified TYH-Hepes media (Free        of glucose and Pyruvate) pH 7.2 to 7.4.    -   6. Leave the sperm to swim out of the tissue for 10 to 15        minutes.    -   7. Then take the 2 ml swim out and centrifuge for 5 minutes at        2000 RPM or subject the sperm to the device disclosed herein        (with or without centrifugation).    -   8. Take the supernatant up to 300 ul or 500 ul.    -   9. Re-suspend up to 1 ml, including 2 ml, with modified        TYH-Hepes (Glucose and Pyruvate Free)    -   10. Wait about 30-40 minutes until sperm stop moving    -   11. Add 1 ml of TYH supplemented with glucose 5 mM and pyruvate        800 uM.    -   12. Centrifuge for 5 minutes at 1500 RPM.    -   13. Take the supernatant up to 500 ul.    -   14. Re-suspend up to 1 ml of TYH supplemented with glucose 5 mM        and pyruvate 800 uM.    -   15. Take 100 ul of the swim out and add it to capacitation media        (TYH supplemented with 15 mM HCO₃ ⁻ and 5 mg/ml serum albumin)        with a final volume of 400 ul.    -   16. Wait about 60 minutes until sperm is fully capacitated (time        can adjusted for species).    -   17. Check motility with CASA system.        Results/Discussion

Proof of principle has been conducted using mouse sperm. This can beextrapolated to other species including farm animals and humans.

Example II—In Vitro Fertilization/Starving Protocol Methods

Day 1:

-   -   Inject females with 5 IU (100 μl) of PMSG at 9-10 p.m. (hormones        were prepared and diluted in sterilized PBS and keep to −20°        C.).        Day 3:    -   Inject females with 5 IU (100 μl) of hCG at 9-10 p.m. (48 h        after PMSG).        Day Before IVF        Media:    -   5 ml TYH-Standard (4 mg/ml BSA) IVF at 37° C., 5% CO₂.    -   8 ml TYH-free glucose and pyruvate (4 mg/ml BSA) for sperm swim        out at 37° C., 5% CO₂.    -   TL-HEPES supplemented with 5% Fetus Bovine Serum prepare the        same of the IVF        For Oocytes:    -   Prepare Tissue Culture dish 35×10 mm with 90 μl of media        TYH-Standard (4 mg/ml BSA) IVF at 37° C., 5% CO₂. See FIG. 2 for        further details.    -   Put different plates into incubator at 37° C., 5% CO₂.        For Oviducts:    -   Prepare Tissue Culture dish 35×10 mm with 90 μl of media        TYH-Standard (4 mg/ml BSA) IVF at 37° C., 5% CO₂.    -   Put different plates into incubator at 37° C., 5% CO₂.        For Sperm:    -   Prepare 2 ml tube of TYH (Free of glucose and pyruvate for sperm        swimming out)    -   Put tube into incubator at 37° C., 5% CO₂.        Day 4:        9:30 Prepare TL Hepes    -   Prepare TL-HEPES supplemented with 5% Fetus Bovine Serum        For oviducts:    -   Prepare dish plate with 2 ml of TL-HEPES supplemented with 5%        Fetus Bovine Serum (one to wash, and other to get the        cumulus-oocyte complex).        10 a.m. Sperm collection    -   Sacrifice male. Sperm cell from the cauda epididymes are spilt        and allowed to swim out in 2 ml TYH-free glucose and pyruvate        and standard TYH control medium for 10 min in 2 ml tube. Place        the tube in at 37° C., 5% CO₂ incubator for 10 min.    -   After 10 min take the 2 ml swim out and centrifuge for 5 minutes        at 2000 RPM.    -   Take the supernatant up to about 300 ul or 500 ul.    -   Re-suspend up to 2 ml with TYH (Glucose and Pyruvate Free) or        standard TYH control at 37° C., 5% CO₂    -   Centrifuge for 5 minutes at 1500 RPM    -   Take the supernatant up to 300 ul or 500 ul.    -   Re-suspend up to 1 ml with TYH (Glucose and Pyruvate Free) or        standard TYH control at 37° C., 5% CO₂    -   Wait until sperm stop moving around 1 hour    -   Add 1 ml of TYH-Standard with glucose and pyruvate at 37° C., 5%        CO₂.    -   Centrifuge for 5 minutes at 1500 RPM    -   Take the supernatant up to 300 ul or 500 ul.    -   Re-suspend up to 500 ul or 1 ml with TYH standard with glucose        and pyruvate at 37° C., 5% CO₂    -   Ready for insemination        10:30-11 a.m. Egg collection while sperm stop moving    -   Sacrifice females super ovulated 13 to 14 hours after hCG        administration.    -   Remove oviducts and place in 1 ml TL-HEPES (5% FBS) medium in        dish plate to rinse of blood and loose tissue.    -   Open the oviducts with thin tweezers, and release the cumulus    -   Using a fine-bore pipette transfer cumulus to a clean 2 ml        TL-HEPES (5% FBS) dish.    -   Transfer cumulus to a clean dish with 3 ml TYH standard at 37°        C., 5% CO₂. Hepes inhibits IVF so make sure wash off the hepes        very well before you place the cumulus in the IVF drop.    -   Transfer cumulus to IVF drop leave at 37° C., 5% CO₂.    -   Ready to be inseminated        11 a.m. Fertilization    -   Inseminate using 20 μl of sperm capacitated. Co-incubate oocytes        and sperm at 37° C., 5% CO₂ for 4 h, and then wash sperm of        oocytes by transferring two times the oocytes into drop 1 and 2        with TYH-Standard with glucose and pyruvate (4 mg/ml BSA) using        a fine-bore pipette.    -   After washing, place oocytes in post-fertilization drop 3 and        incubate up to 24 h at 37° C., 5% CO₂.        Day 5:    -   12:30 a.m. to 2:00 pm Putative zygote evaluation:    -   Check for two pronuclei or two embryo    -   Follow embryo culture protocol

Embryo Culture

-   -   Following day after IVF Prepare dishes with KSOM medium drops        (50 ul) covered with light mineral oil and put it in CO₂        incubator at 37 C for 1 hour before transferring 2 cell stage        embryos.    -   Transfer 2 cell embryos to KSOM medium (wash 2 times) make the        same dish as the IVF, instead add 25 ul of KSOM medium.    -   Transfer only 35 2-cell embryos per drop of KSOM culture drop    -   This day follow the pseudo-pregnant female preparation chart.    -   Wait 2.5 days until blastocyst formation; see FIG. 3.    -   At blastocyst stage ready to transfer        Embryo Transfer and Pseudo-Pregnant Females

DAY-0 DAY-1 DAY 2 DAY 3.5 IVF start 11 AM Transfer 2 cell Do embryoFinish 4 pm Embryos to KSOM transfer before (1 pm to 4 pm) noon in theMate the females Check plugs at 9 12 pm 2.5 day with vasectomized am,and separate Males at 5 pm. females with plug Mice mate at 12 pm (day1)midnight (day 0)

Thursday Day-0 Friday Day-1 Saturday Day-2 Sunday-Day3 Monday 3.5 IVFstart 11 Transfer 2 cell Check for Do embryo AM Embryos to KSOM Morulastransfer at 6 to Finish 4 pm (1 pm to 4 pm) 12 am am in the morningRecipients Mate the females with Check plugs at 9 12 pm (day 2) 12 pm2.5 day Females- vasectomized Males at am, and separate 5 pm. Mice mateat females with midnight 12 pm (day 0) plug 12 pm (day1)Embryo Transfer Procedures1. Place a 15 μl drop of culture medium (KSOM already equilibrated at 37C 5% CO₂) onto the lid of a 100 mm petri dish (Falcon 1029, or similar).2. Load 12-20 blastocysts into the medium using a standardembryo-handling pipette. (Note: optimal number of embryos to transferwill vary depending upon mouse strain and manipulations embryos havereceived.)3. Place the NSET device onto a P2 pipette that has been set to 1.8 μl.Recommended pipettes are the Pipette Rainin Classic PR2, 0.1-2 μl orGilson Pipetman P2, 0.2-2 μl.4. Press pipette plunger to first stop, lower tip of the NSET deviceinto medium and slowly pull embryos into the tip. Remove NSET device tipfrom medium.5. Carefully set pipette to 2.0 μl to create a small air bubble at NSETtip to help ensure embryos stay inside device tip during insertion intothe mouse. Gently lay pipette with loaded tip aside (near cage) for usein step #96. Place the un-anesthetized recipient female on top of a cage with awire rack, allowing the mouse to “grab” the cage bar surface with itsforefeet. Grasp the midpoint of the tail using thumb and forefinger, andangle the tail upward while lightly pressing the base of the tail withthe opposite edge of the hand.7. Gently place smaller speculum into mouse's vagina, and then remove.This will help open the vagina.8. Place larger speculum into vagina. Using an adequate light source,shine the light into the speculum to visualize the cervix.9. While holding the female mouse with one hand as described in step #6,carefully pick up the pipette and gently insert the NSET device tip intothe large speculum and through the cervix. Once NSET device hub contactsspeculum, expel embryos by pressing plunger completely.10. Gently remove NSET device without releasing pipette plunger andremove speculum. Return mouse to cage. No post-procedure monitoring isrequired.Artificial InseminationAnimals: Female mice (at least 8 weeks old); Male mice as sperm donorsSperm (C57BL6/J);Male vasectomized mice (VASEX=vasectomized male)Equipment: NSET device with specula; P-20 Rainin/Gilson pipette; 1 ccsyringes, 26 gauge needles; Scissors, forceps; IVF Tissue culture dishes(Falcon Cat#353653); Microscope (s); Wire-topped cage

Monday Tuesday Wed Day-0 Day-1 Day-2 Thursday-Day3 Friday 4 PMSG NONEhCG AI at 9:00 am Injection Injection Add 40 ul of 5 IU 5 IUsperm/female 5:30 pm 5:00 pm Recipients NONE Put super-ovulated Checkplugs at Females- females with the 9 am, and separate vasectomized malesfemales with plugSperm Preparation1. Take one male Mice, sacrifice via dislocation or CO2 chamber.2. Open the abdomen with fine scissors. Begin from the pelvic area andmake a V shape so you can see all the organs3. Look for the testis (white pale balls) and follow the seminiferoustubules until you find the cauda of the epididymis (looks like a smallbrain).4. Take the cauda epididymis and make three or four incisions until yousee white fluid coming out.5. Take one epididymis for treatment and one for the control6. Place epididymis in 2 ml modified TYH-Hepes media with 5% BSA (Freeof glucose and Pyruvate) pH 7.2 to 7.4. Notice the control must haveglucose and pyruvate.7. Leave the sperm to swim out of the tissue for 10 to 15 minutes.8. Then take the 2 ml swim out and centrifuge for 5 minutes at 2000 RPM.9. Take the supernatant up to 300 ul.10. Re-suspend up to 2 ml with modified TYH-Hepes media with 5% BSA(Glucose and Pyruvate Free)11. Then take the 2 ml swim out and centrifuge for 5 minutes at 1500 RPM12. Take the supernatant up to 300 ul.13. Wait around 40 minutes until sperm stop moving14. Ready to inseminate the female: At 9:00 am: Deliver sperm to theuterine horn using the NSET procedure.

-   -   Place the NSET device onto a P-20 pipette that has been set to        20 pl.    -   Press pipette plunger to first stop, lower tip into media at the        edge of the sperm sample and slowly load sperm into the NSET        device. Avoid clumps. Set aside pipette. Sperm at the edge of        the sperm sample are    -   Place the un-anesthetized recipient female on the top of a cage,        allowing the mouse to “grab” the cage bar surface with its        forefeet. Grasp the midpoint of the tail using thumb and        forefinger, and angle the tail upward while lightly pressing the        base of the tail.    -   Place small speculum into vagina.    -   While holding the female mouse with one hand as described above,        carefully pick up the pipette and insert the NSET tip into the        speculum, through the cervix and into the uterus. Once NSET hub        contacts speculum, expel sperm by pressing plunger to the first        stop.    -   Repeat procedure to deliver a total sperm volume of 40 but wash        NSET device with a TYH-Hepes media with 5% BSA (Free of glucose        and Pyruvate) every time device goes into the uterus. This        prevents contamination of Starved sperm with metabolic        substrates present in the uterus    -   Remove NSET device and speculum. No post-procedure monitoring is        required.        15. Immediately pair the female with a VASEX male overnight.        Copulatory activity seems to be required to obtain pups from        this procedure but not for embryo fertilization.        2. Dissolve Mating Pairs Day 5:        a. Remove the female from the VASEX male cage.        b. Visually check for a copulation plug. The female is removed        from the mating cage and transferred to the top of a wire-topped        cage. Visual inspection and/or a blunt-end probe may be used to        determine the presence of a vaginal plug (FIG. 4).        Results

Example III—Method for Treating Sperm with Ca²⁺ Ionophores Alone and inCombination with Starvation to Improve Embryo Development

Abstract

Mammalian sperm acquire fertilizing capacity in the female tract in aprocess called capacitation. As part of capacitation, sperm undergochanges in their motility pattern (i.e., hyperactivate) and becomeprepared for an exocytotic acrosome reaction that is necessary forfertilization. At the molecular level, capacitation requires a fastactivation of protein kinase A (PKA) which is followed byhyperpolarization of the sperm plasma membrane and an increase inintracellular Ca²⁺. Genetic or pharmacological inhibition of thesepathways results in loss of fertilizing ability both in vivo and invitro. Recently, it was demonstrated that transient incubation of mousesperm with the Ca2+ ionophore A23187 accelerated capacitation andrescued fertilizing capacity in sperm with inactivated PKA function (1).Based upon these results, it was believed that A23187 could be used toovercome defects in signaling pathways upstream of the increase inintracellular Ca²⁺ required for capacitation. It is herein shown that apulse of ionophore induces fertilizing capacity in sperm from infertileCatSper1 (sperm specific Ca²⁺ channel), Adcy10 (soluble adenylyl cyclasesAC) and SLO3 (sperm-specific K+ channel) KO mice. In contrast, spermfrom infertile mice lacking the Ca²⁺ efflux pump PMACA4 (Plasma membraneCa²⁺-ATPase) were not rescued by ionophore. These results indicate thata transient increase in intracellular Ca²⁺ can be used to overcomegenetic infertility in mice and suggest this approach may proveadaptable to rescue male infertility of other species in which in vitrofertilization protocols are currently unsuccessful.

Introduction

While treating sperm with Ca²⁺ ionophores is known (Ca²⁺ ionophoreA23187 can make mouse spermatozoa capable of fertilizing in vitrowithout activation of cAMP-dependent phosphorylation pathways). It wasnot appreciated that this treatment impacted embryo development.Further, Ca²⁺ ionophore treatment (such as Ca²⁺ ionophore A23187) hasnot been used in conjunction with starvation to improve fertilityprocedures.

In 1978, Steptoe and Edwards reported the birth of Louise Joy Brown, thefirst successful “Test-Tube” baby (2). A major step toward thisachievement (3) occurred in the early 1950's, when Chang (4) and Austin(5) demonstrated independently that sperm have to be in the femalereproductive tract for a period of time before acquiring fertilizingcapacity, a phenomenon now known as sperm capacitation. Capacitationincludes all post-ejaculation biochemical and physiological changes thatrender mammalian sperm able to fertilize (4, 5). As part ofcapacitation, sperm become prepared to undergo acrosomal exocytosis (6,7) and undergo changes in their motility pattern (e.g. hyperactivation).Although the molecular basis of these physiological processes is notwell understood, capacitation is associated with: 1) activation of acAMP/protein kinase A (PKA) pathway (8, 9); 2) loss of cholesterol (10,11) and other lipid modifications (12); 3) increase in intracellular pH(pHi) (13); 4) hyperpolarization of the sperm plasma membrane potential(14, 15, 16); 5) increase in intracellular Ca²⁺ concentration [Ca²⁺]i(17); and 6) increase in protein tyrosine phosphorylation (9, 18). Thesepathways were first identified as playing a role in capacitation usingcompounds that stimulate or block the respective signaling processes.More recently, the essential role of cAMP, Ca²⁺ and plasma membranehyperpolarization was confirmed using KO genetic approaches (19, 20).

The role of cAMP in capacitation and fertilization was asserted usingreagents such as cAMP agonists (dibutyryl cAMP, 8-BrcAMP) andantagonists of PKA-dependent pathways (e.g. H89, PKI, rpScAMP), as wellas other conditions in which soluble adenylyl cyclase Adcy (10 21), themajor source of cAMP in sperm, cannot be activated (e.g. HCO₃ ⁻-freeincubation media; addition of KH7, a specific sAC inhibitor) (for reviewsee 7). The roles of cAMP were confirmed using KO genetic mouse modelslacking either the PKA sperm-specific catalytic splicing variant Cα2, orsAC; these mice are sterile and their sperm cannot fertilize in vitro(22). It was demonstrated that hyperpolarizing changes in membranepotential are necessary and sufficient to prepare the sperm for aphysiological acrosome reaction (23). Accordingly, sperm missing thesperm-specific K⁺ channel SLO3 cannot hyperpolarize and are infertile(24, 25). Finally, Ca²⁺ was shown to be essential for hyperactivationand the acrosome reaction by using Ca²⁺-free incubation media with orwithout addition of chelating agents such as EGTA to decrease this ionconcentration or using Ca²⁺ ionophores such as A23187 to elevate it (1).Consistent with this, sperm-specific Ca²⁺ channel complex CatSper KOmice are infertile, and their sperm are unable to hyperactivate.

Recently, it was found that addition of Ca²⁺ ionophore A23187 produced afast increase in intracellular Ca²⁺ that was accompanied by completeloss of sperm motility (1). After A23187 removal, intracellular Ca²⁺levels dropped and sperm gain hyperactive motility (1). In addition toinducing hyperactivated motility, this Ca²⁺ ionophore A23187 pulseenhanced fertilizing capacity. Interestingly, this Ca²⁺ ionophore pulsesupported capacitation in sperm incubated under non-capacitatingconditions, and it induced hyperactivation and the capacity to fertilizein vitro even under conditions where cAMP-dependent pathways are blocked(1). These results suggested that A23187 could overcome defects in thesignaling pathways upstream of the increase in intracellular Ca²⁺required for capacitation. This was tested using infertile genetic mousemodels. Consistent with the hypothesis, a short A23187 pulse overcomesthe infertile phenotypes of CatSper (19), sAC (22) and SLO3 KO sperm(25). The previous results suggested that subsequent washout of A23187,sperm intrinsic mechanisms involved in extruding Ca²⁺ are necessary toinduce hyperactivation and fertilizing capacity (1). Consistent withthis hypothesis, sperm lacking the Ca²⁺ efflux pump PMCA4, whichmediates Ca²⁺ extrusion (26), were not rescued by the ionophoretreatment, suggesting that this ATPase is required downstream to removeexcess intracellular Ca²⁺.

Materials and Methods

Materials

Different materials and chemicals were purchased from differentcompanies (codes between parenthesis represent the catalog number of therespective compound): Calcium Ionophore A23187 (C7522; dissolved in DMSO2 mM stock), Bovine serum albumin (BSA, fatty acid-free) (A0281),Tween-20 (P7949), fish skin gelatin (G7765), Pregnant mare serumgonadotropin (G4877) and human chorionic gonadotropin (CG5), werepurchased from Sigma (St. Louis, Mo.). Non-Surgical Embryo Transfer(NSET) Device was acquired from Paratechs (Billerica, Mass.).N-[2-[[3-(4-bromophenyl)-2-propen-1-yl]amino]ethyl]5-isoquinolinesulfonamide,and dihydrochloride H-89 (130964-39-5) were purchased from Caymanchemical (Ann Arbor, Mich.). Anti-phosphotyrosine (anti-PY) monoclonalantibody (clone4G10), embryo transfer light mineral oil (ES-005-C) andEmbryoMax® KSOM Medium (1×) w/ 1/2 Amino Acids (MR-106-D) were obtainedfrom Millipore (Billerica, Mass.). Rabbit monoclonal anti-phosphoPKAsubstrates (anti-pPKAS) (clone100G7E), was purchased from Cell Signaling(Danvers, Mass.). Horseradish peroxidase-conjugated anti-mouse andanti-rabbit IgGs were purchased from Jackson ImmunoResearch Laboratories(West Grove, Pa.) and GE Life Sciences. Triton X-100 (161-0407), 30%Acrylamide and β-Mercaptoethanol was obtained from Biorad.

Animals

CD1 (Charles River Laboratories, Wilmington, Mass.) and C57BL/6background mice, 7-18 wk of age, were used. Infertile KO mice geneticmodels (CatSper−/−, sAC−/− and SLO3−/−) were in C57BL/6 background;PMCA4−/− mice were in FVBN background. For CatSper embryo recipients,surrogate mothers were CD1 females, 8-12 wk of age. Animals weresacrificed in accordance with the Animal Care and Use Committeeguidelines of UMass, Amherst. In experiments in which phosphorylation byPKA and tyrosine phosphorylation was investigated, CD1 and C57BL/6 malemice were used as indicated in the respective figure legend.

Media

Medium used for sperm capacitation and fertilization assays wasToyoda-Yokoyama-Hosi (standard TYH) medium. Containing 119.37 mM NaCl,4.7 mM KCl, 1.71 mM CaCl₂.2H₂O, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄. 7H2O, 25.1mM NaHCO₃, 0.51 mM Na-pyruvate, 5.56 mM glucose, and 4 mg/mL bovineserum albumin (BSA), 10 μg/mL Gentamicin and phenol red 0.0006% at pH7.4 when equilibrated with 5% CO₂. To analyze the role of capacitationin phosphorylation pathways an HEPES-modified TYH media was used. Mediathat does not support capacitation (Non-Cap) contained 20 mM HEPESinstead of HCO3− and does not contain BSA. For capacitating conditionsHEPES-modified TYH was supplemented with 15 mM HCO3⁻ and 4 mg/ml of BSA.Ca²⁺ ionophore A23187 (Sigma Aldrich, location) was used at 20 μM in TYHor H-TYH. Non-capacitating H-TYH was prepared by replacing 25 mM NaHCO₃with 20 mM Na-Hepes. Day before IVF add 4 mg/ml BSA.

Mouse Sperm Preparation for Western Blots

Cauda epididymal mouse sperm were placed in 1 ml Hepes-TYH media asstated in figure legend for each experiment. After 10 min incubation at37° C., epididymides were removed, and the suspension adjusted withnon-cap medium to a final concentration of 1-2×10⁷ cells/ml. Afterdilution 1:4 (total 400 ul), sperm were incubated at 37° C. for 1 hourin conditions that support or not capacitation. For capacitation, mediawas supplemented with 15 mM NaHCO₃ and 4 mg/ml BSA. For A23187treatment, 500 uL of the sperm suspension were taken from the initialswim out and supplemented with 20 μM A23187 for 10 minutes. Then, A23187was washed off with 2 rounds of centrifugations 1-) 2000 RPMS and the2-) 1500 RPMS for 5 min each. Sperm were re-suspended in free A23187media. To evaluate the behavior of A23187 when PKA is inactivated, H89was used at a concentration of 50 uM for all incubation periodsincluding those used for washing A23187. Sperm proteins were thenextracted for western blotting.

SDS-PAGE and Immunoblotting

Sperm were centrifuged, and washed in 1 ml of phosphate buffer solution(PBS), re-suspended in Laemmli sample buffer (63), and boiled for 4 min.Before Loading, 5% β-mercaptoethanol was added to the protein extractsand boiled for 3 min. Protein extracts equivalent to 1×10⁶ sperm wereloaded per line and subjected to SDS-PAGE an electro-transfer to PVDFmembranes (Bio-Rad) at 250 mA for 60 min on ice. For anti pPKAsubstrates Western blots, membranes were blocked with 5% fat-free milkin TBS containing 0.1% Tween 20 (T-TBS). For anti-pY, membranes wereblocked with 20% fish skin gelatin (54) in T-TBS. Antibodies werediluted in TBS containing 0.1% Tween-20 as follows: 1/10,000 for anti-PY(clone4G10), and 1/1000 for anti-pPKA (clone100G7E). Secondaryantibodies were diluted 1/10,000 in T-TBS and developed using anenhanced chemiluminescence detection kit (ECLplus, Amersham, GEHealthcare) according to the manufacturer's instructions. Whennecessary, PVDF membranes were stripped at 65° C. for 15 min in 2% SDS,0.74% β-mercaptoethanol, 62.5 mM Tris, pH 6.5, and washed six times for5 min each in T-TBS.

Motility and IVF Sperm Ionophore Pre-Treatment

Sperm from CD-1, C57BL6 (K.O control), CatSper KO 19, SLO3 KO (56),Soluble Adenylyl Cyclase KO (20), and PMC4 KO (35) cauda epididymideswere allowed to swim out in 1 mL of standard TYH for 10 minutes. Eachswim out tube was split in two halves (500 ul each) and one half wasincubated with 20 uM A23187 for 10 minutes. Then, the A23187 was washedby centrifugation as described above (2000 and 1500 RPMS×5 min), theremaining sperm were re-suspended in free A23187 TYH standard medium andcapacitated for an addition 1 hour and 20 minutes before adding thesperm to the fertilization drop or for CASA analysis.

Sperm Motility Analysis

Sperm suspensions (25 μl) were loaded into one pre-warmed chamber slide(depth, 100 μm) (Leja slide, Spectrum Technologies) and placed on amicroscope stage at 37° C. Sperm movements were examined using the CEROScomputer-assisted semen analysis (CASA) system (Hamilton ThorneResearch, Beverly, Mass.). The default settings include the following:frames acquired: 90; frame rate: 60 Hz; minimum cell size: 4 pixels;static head size: 0.13-2.43; static head intensity: 0.10-1.52; statichead elongation: 5-100. Sperm with hyper activated motility, defined asmotility with high amplitude thrashing patterns and short distance oftravel, were sorted using the criteria established by (64). The data wasanalyzed using the CASA nova software (64). At least 20 microscopyfields corresponding to a minimum of 200 sperm were analyzed in eachexperiment.

Video Recordings

Sperm suspensions (25 μl) were loaded into one pre-warmed chamber slide(depth, 100 μm) (Leja slide, Spectrum Technologies). Videos wererecorded for 15 seconds using an Andor Zyla microscope camera (Belfast,Northern Ireland) mounted on Nikon TE300 inverted microscope (Chiyoda,Tokyo, Japan) fitted with 10 and 20 times objective lenses. Sampletemperatures were maintained at 37° C. using a Warm Stage (Frank E.Fryer scientific instruments, Carpentersville, Ill.).

Mouse Eggs Collection and IVF Assays

Metaphase II-arrested eggs were collected from 6-8 week-old superovulated CD-1 female mice (Charles River Laboratories). Females wereeach injected with 5-10 IU equine chorionic gonadotropin and 5-10 IUhuman chorionic gonadotropin 48 h apart. The cumulus-oocyte complexes(COC's) were placed into a well with 500 μl of media (TYH standardmedium) previously equilibrated in an incubator with 5% CO₂ at 37° C.Fertilization wells containing 20-30 eggs were inseminated with sperm(final concentration of 2.5×10⁶ cells/ml) that had been incubated for 1h and 20 min (in a medium supporting capacitation with or withoutcalcium ionophore treatment A23187). After 4 h of insemination, eggswere washed and put in a fresh media. The eggs were evaluated 24 hpost-insemination. To assess fertilization the three following criteriawere considered: 1) the formation of the male and female pronuclei, 2)the emission of the second polar body, and 3) two cells stages.

Embryo Culture, Embryo Transfer and Mice Genotyping

Fertilized 2 cell embryos were cultured in KSOM media to blastocyststage between 3.5 days wt and K.O between 3.8 to 4.1 days. Then theywere transferred to 2.5 days post coitum (dpc) pseudo-pregnant CD-1recipient females using the non-surgical uterine embryo transfer device(65). Pseudo-pregnant CD-1 recipient females were obtained by matingwith vasectomized males one day after in vitro fertilization. Onlyfemales with a clear plug were chosen as embryo recipients; late morulaand early stage blastocysts were chosen to be transferred. Routinegenotyping was performed with total DNA from tail biopsy samples fromweaning age pups as templates for PCR using genotyping primers forCatsper gene forward [5′-TAAGGACAGTGACCCCAAGG-3′] and reverse[5′-TAAGGACAGTGACCCCAAGG-3′] and for the reporter gene Lacz forward [5′TGATTAGCGCCGTGGCCTGATTCATTC-3′] and reverse[5′-AGCATCATCCTCTGCATGGTCAGGTC-3′] (19).

Results

A23187 Improves Hyperactivation and Fertilizing Capacity of Sperm fromC57BL6 Mice.

The relevance of genetic background for sperm physiology and for theirability to fertilize in vitro has been well-established (27, 28, 29,30). Over the years, C57BL6 has been a common genetic background forstudying KO genetic mouse models. Unfortunately, relative to sperm frommice of other genetic backgrounds, specifically CD1 mice, sperm fromC57BL6 exhibit significantly lower hyperactivation rates whencapacitated (FIG. 5 A) and are less efficient for in vitro fertilization(31) (FIG. 5B). To test the effect of a short pulse of Ca²⁺ ionophore,sperm from CD1 was compared with those from C57BL6 mice. A23187treatment increased the percentage of hyperactive C57BL6 sperm tosimilar levels as those obtained using CD1 sperm (FIG. 5 A). Moreover,this increase was followed by a significant increase in C57BL6 spermfertilization rate (FIG. 5B). Two-cell derived from the use of controlsperm developed to blastocysts in about 50%. This number is expected forsperm derived from this mouse strain. Surprisingly, after A23187treatment, over 80% of fertilized eggs continued to the blastocyst stage(FIGS. 5C and D), and when non-surgically transferred to pseudo pregnantmice females, became live pups. Capacitation requires PKA activation(32), and, as expected, in the presence of the PKA inhibitor H89, C57BL6sperm are unable to fertilize in vitro and do not show the prototypicalincrease in PKA substrate phosphorylation (FIGS. 5E and F). Remarkably,as seen previously with CD1 sperm, incubating H89-treated C57BL6 spermfor 10 min in A23187 was sufficient to induce fertilizing capacity (FIG.5E), despite the fact that PKA remains inactive (FIG. 5F). These datashow that transient exposure to A23187 can improve IVF success forC57BL6 mice strains and suggest this treatment has the potential tofacilitate distribution of C57BL6 mouse lines.

A₂₃₁₈₇ Treatment Induced Hyperactivation and Fertilizing Capacity ofCatSper1 KO Sperm.

In the absence of the CatSper channel complex, sperm fail to undergohyperactivated motility and are unable to fertilize (19). To test theextent by which Ca²⁺ ionophore treatment can overcome the CatSperinfertile phenotype, sperm from CatSper1 KO mice were incubated inconditions that support capacitation in the absence or in the presenceof 20 μM A23187. After 10 min the sperm were washed twice bycentrifugation in A23187-free media and the percentage of hyperactivesperm was measured using CASA. As expected, in the absence of A23187,CatSper KO sperm did not undergo hyperactivation (FIG. 6 A). However,once exposed to Ca²⁺ ionophore, a significant number of CatSper KO spermexhibited hyperactivated motility (FIG. 6 A). In addition,A23187-treated CatSper KO sperm were competent to fertilize metaphaseII-arrested eggs in vitro (FIG. 6B). A fraction of the fertilized eggsthat reached blastocyst stage were non-surgically transferred topseudopregnant female mice (33), and five CatSper (+/−) mouse pups wereborn from two different females (FIG. 6C). These heterozygous F1 micewere fertile, as mating a male and female from this heterozygouspopulation yielded a normal litter with 1 wild type, 4 heterozygous and3 CatSper KO F2 progeny (FIG. 6D).

A₂₃₁₈₇ Treatment Also Rescued Fertilizing Capacity in Sperm of sAC KOand SLO3 KO Mice.

Among the earliest molecular events during capacitation areup-regulation of cAMP-dependent pathways (32) and hyperpolarization ofthe sperm plasma membrane (23). These events precede the essentialincrease in intracellular Ca²⁺, and it was tested whether defects ineach of these cascades can be rescued by Ca²⁺ ionophore pulse. As shownabove, a short pulse of Ca²⁺ ionophore bypassed the need for thecapacitation-induced PKA activation (1) (FIGS. 5E and F). Under normalcapacitation conditions, sAC KO sperm are almost immotile (FIG. 7A),while SLO3 KO sperm are able to move. However, neither sAC KO nor SLO3KO sperm have the ability to undergo hyperactivation (FIG. 7B). Despitethis phenotype, once treated with A23187 for 10 min, a significantfraction of sAC KO sperm became motile and both sAC KO and SLO3 KO spermunderwent hyperactivation (FIG. 7B). Moreover, A23187 treatment inducedfertilizing capacity in sperm from both KO models (FIG. 7C).

A₂₃₁₈₇ Treatment does not Rescue Pmca4 KO Sperm.

It was previously shown that the increase in intracellular Ca²⁺ causedby A23187 has to be followed by a reduction of intracellular calciumafter removal of the ionophore (1). In sperm, two molecules are thoughtto mediate Ca²⁺ extrusion, namely the Na⁺/Ca²⁺ exchanger and the moreefficient sperm-specific Ca²⁺ ATPase PMCA4 (34) Male PMCA4 KO mice areinfertile (35), suggesting this molecule is involved in regulation ofCa²⁺ homeostasis in sperm. It was hypothesized that sperm lacking PMCA4would not respond to A23187 rescue. PMCA4 KO sperm display poor motilityand do not hyperactivate (FIGS. 7 A and B). Addition of A23187 renderedall the sperm motionless and their motility was not recovered afterionophore removal (FIG. 7D). Consequently, neither their hyperactivatedmotility nor their fertilizing capacity was rescued (FIG. 7E).

Discussion

Capacitation encompasses a series of sequential and concomitantbiochemical changes required for sperm to gain full fertilizationcompetency. Despite the relevance of capacitation, the molecularmechanisms intrinsic to this process are not well understood. A veryearly event in sperm capacitation is the activation of motility by acAMP pathway (36). The activation of cAMP synthesis occurs immediatelyafter sperm are released from the epididymis and come into contact withhigh HCO₃ ⁻ and Ca²⁺ present in the seminal fluid (37, 38). Plasmamembrane transport of these ions regulates sperm cAMP metabolism throughstimulation of Adcy10 (aka sAC) (21 20, 39). sAC activation, elevatesintracellular cAMP and stimulates PKA. Then, PKA phosphorylates targetproteins which initiate several signaling pathways. These pathwaysinclude a sperm plasma membrane hyperpolarization, an increase in pHi,and an increase in intracellular Ca²⁺ ions. Consistent with theinfluence of these events, KO mice genetic models in which any of thesepathways is interrupted are infertile.

Physiologically, sperm capacitation is associated with changes in theirmotility pattern collectively known as hyperactivation and with thepreparation for a physiological acrosome reaction. Originally observedin hamster sperm moving in the oviduct, hyperactivated motility (40) waslater described in other mammalian species including humans (41).Hyperactivation is associated with a strong high-amplitude asymmetricalflagellar beating that appears to be essential for the sperm to loosentheir attachment to the oviductal epithelium and to penetrate the zonapellucida (42). Consistent with an essential role of hyperactivation forfertilization competency, one of the most common phenotypes observed insperm from many different infertile knock-out models including thoseused in the present work (e.g. CatSper, sAC, SLO3 and PMCA4) is lowmotility and/or defects in hyperactivation (22, 32, 38, 43, 44, 45).

Although very little is known about the molecular pathways regulatinghyperactivation, Ca²⁺ ions have been shown to play roles in theinitiation and maintenance of this type of movement (46, 47, 48, 49).Most of the information regarding the role of Ca²⁺ in hyperactivationhas been obtained using loss of function approaches analyzing spermmotility in media devoid of Ca²⁺ ions. Gain of function experimentsusing Ca²⁺ ionophores (e.g. A23187, ionomycin) to increase [Ca²⁺]i haveyielded unexpected results because, instead of enhancinghyperactivation, these compounds stopped sperm movement (50, 51, 52).Despite being motionless, ionophore-treated sperm are alive as theyrecover motility after the compound is quenched with lipophilic agents(50) or washed by centrifugation (52). The reversibility of the A23187effect suggests that the sperm is able to return to physiological[Ca²⁺]i after a drop in free ionophore concentration. In previous work,it was shown that a short incubation period with A23187, in addition toinitiating hyperactivation, accelerated the acquisition of fertilizingcapacity. Unexpectantly, the data indicated that 10 min incubation withA23187 followed by wash out induced fertilization competence withoutactivation of cAMP-dependent signaling pathways that are needed forcapacitation (1).

Considering these results, it was hypothesized that a temporaryelevation of intracellular Ca²⁺ primes the sperm for hyperactivation andbypasses the need for other signaling pathways required to up-regulateCa²⁺ influx in sperm. To test this hypothesis, in the present work, fourKO models affecting independent signaling pathways were selected. Threeof these signaling molecules are believed to act upstream of theincrease in Ca²⁺ required for hyperactivation: CatSper, sAC and SLO3.Sperm from each of these mouse models are unable to undergohyperactivation and are incapable of fertilizing metaphase II arrestedeggs in vitro. In addition, PMCA4b KO sperm were used, which would notallow intracellular Ca²⁺ lowering after flooding with this ion. Spermfrom PMCA4b KO mice are deficient in both progressive and hyperactivatedmotility resulting in sterility (53, 26). PMCA4 has been shown to be theprincipal source of Ca²⁺ clearance in sperm and it is essential toachieve a low resting [Ca²⁺]i (34). Consistent with the hypotheses, ashort incubation of sperm with A23187 induced hyperactivation ofCatSper, sAC and SLO3 KO but not of PMCA4 KO sperm.

Male factors contribute to approximately half of all cases ofinfertility (54, 55). However, in over 75% of these cases it is unusualto have a clear diagnosis of the abnormalities found in semenparameters. Currently, assisted reproductive technologies (ART) remainthe main therapy available. Recent studies using KO mouse models,including those used in the present work, revealed that loss of functionof a variety of genes results in infertility. Interestingly, several ofthese models present normal sperm counts and their main deficiency isfound in capacitation-associated processes such as impediments tohyperactivate (19), to undergo the acrosome reaction (56), or to gothrough the utero-tubal junction in vivo (57, 58). It was hypothesizethat strategies designed to elevate [Ca²⁺]i such as the use of A23187pulse denoted above should overcome the need of upstream signalingpathways including but not limited to PKA activation. In addition,although IVF has been successfully employed in multiple species (6),requirements of sperm for capacitation vary greatly among species andhave been developed for each sperm type essentially by trial and error.In some species, such as the horse, effective methods for IVF have stillnot been established despite decades of work (59). Failure of equine IVFdoes not appear to be associated with oocyte characteristics (60), butis associated with the inability of horse sperm to hyperactivate and topenetrate the egg zona pellucida (ZP) (61), two landmarks ofcapacitation. A better understanding of capacitation signaling processeshave the potential to generate “universal” IVF technology that can beused in endangered/exotic species for which ART is not currentlyavailable.

Improving IVF conditions would be of great value; however, at theclinical level, ICSI has replaced IVF when confronted with cases ofunknown male factor infertility. ICSI is reliable and from the patient'spoint of view more economical because of higher probability of success.Despite these advantages, ICSI bypasses certain aspects of normalfertilization and may bear effects that are not easily observed (e.g.epigenetic alterations). Taking this into consideration, a method toimprove IVF can be a desirable option in some male factor cases. It isworth noting that A23187 has already been used in the clinic forpatients with repeated ICSI failure (62). In these cases, eggs aretransiently incubated with ionophore after ICSI, which exposes thezygote to high Ca²⁺. On the contrary, when sperm are treated withA23187, the ionophore is washed and does not come in contact with theembryo. More interestingly, overcoming infertility problems related tomotility and hyperactivation could have other potential uses in theclinic. For example, this methodology could be used to improve thesuccess rate of intrauterine insemination which is a significantly lessinvasive and less costly procedure than either IVF or ICSI.

Example IV—Mouse Sperm

It is well known that out-breed and in-breed mice sperm differ in theability to capacitate and fertilize the egg. According to the NationalInstitute of Health (NIH-US) almost 90% of research is done in in-breedmice and the most common breed used is C57BL6. Wild type C57BL6 micehave shown low fertility in vivo compared to out-breed strains. Inaddition, when it comes to sperm in vitro hyperactivation andfertilization, fertility is also reduced. Therefore sperm from C57BL6mice are a good model to show if a particular treatment can improvefertilization parameters (Navarrete et al., Sci. Rep. 2016,demonstrating that a transient exposure to calcium ionophore A23187improves hyperactivation and fertilizing capacity of sperm from C57BL6/Jmice in vitro. In the experiments presented below, C57BL6 in-breed micestrain was used.

Sperm Cell Signaling Cascades and Protein Phosphorylation:

After ejaculation, mammalian sperm are not able to fertilize, theyrequire being in the female tract for a certain period of time which isspecies-specific. The physiological changes that occur to the spermduring this time period are collectively known as capacitation. Spermcapacitation can be mimicked in vitro in defined media containing: 1)ions such as Na+, Cl−, HCO3−, Ca2+ and Mg2+; 2) energy sources such asglucose, pyruvate, lactate or others; 3) a cholesterol acceptor such asbovine serum albumin (BSA) or beta-cyclodextrin. The in vitrocapacitation media is used to incubate mammalian sperm before combiningthem with eggs during in vitro fertilization. Capacitation is associatedwith changes in the motility pattern. After incubation in capacitationmedia, sperm undergo changes in their movement known as hyperactivation.Hyperactivation motility has been associated with the sperm ability tofertilize.

At the molecular level, capacitation is associated with a fast increasein cAMP, mediated by the atypical adenylyl cyclase Adcy10, followed bythe activation of cAMP-dependent kinase, PKA. This increase in PKA isdependent on the presence of HCO3− and BSA in the capacitation media andcan be measured using anti phospho antibodies against a PKA-consensusphosphorylation sequence RXXS (where X is any type of aminoacid) (FIG.8A). Downstream of the activation of PKA, there is an increase intyrosine phosphorylation which can be measured using antiphosphotyrosine antibodies (FIG. 8B). As shown in the figures, neitherPKA activation nor the increase in tyrosine phosphorylation occur in theabsence of HCO3− and BSA (lane 1 in both panels A and B). After addingHCO3− and BSA, phosphorylation patterns are activated (lane 2 in bothpanels A and B). In both cases, sperm are incubated in the presence ofthe energy nutrients glucose (5 mM) and pyruvate (0.5 mM).

The experiments depicted in FIGS. 8 A and B were performed to evaluatehow the different substrates affect phosphorylation pathways. Whileglucose produces energy by glycolysis and might be also coupled tooxidative phosphorylation through the use of pyruvate and lactate at theend of glycolysis. Pyruvate can only be used by the mitochondria. It isshown that PKA activation occurs with both type of substrates (Lane 3and 4 in FIG. 8A). On the other hand, the increase in tyrosinephosphorylation occurs normally with glucose as substrate (lane 3 inFIG. 8B), but it is reduced when only pyruvate is present in theincubation media (lane 4 in FIG. 8B). When sperm are incubated for 1hour in the absence of energy nutrients, PKA activity and the increasein tyrosine phosphorylation are not observed (lane 5 in both FIGS. 8 Aand B). Addition of glucose, pyruvate or both after 1 hour incubation inthe absence of nutrients rescued activation of PKA (lanes 6, 7 and 8 inFIGS. 8 A and B); on the other hand only when glucose was present theincrease in tyrosine phosphorylation was observed (lanes 6, 7 and 8 inFIGS. 8 A and B).

Aliquots of sperm treated as described for the phosphorylation assays inFIGS. 8 A and B, were evaluated for motility using Computer AssistedSperm Analysis (CASA). It was observed that in the absence of nutrientsfor 1 hour, the percentage of motile sperm was zero. However, themotility was rescued by the addition of glucose, pyruvate or both (FIG.8C). As mentioned above, hyperactivated sperm motility increased whensperm are incubated in capacitation conditions (presence of HCO3− andBSA), compare Bar 1 with Bar 2 in FIG. 8D. In the absence of nutrients(Bar 5 in FIGS. 8C and D), no motility is observed, and therefore, thepercentage of hyperactivated sperm is also zero. When nutrients areadded, hyperactivation is rescued (Bars 6, 7 and 8 in FIG. 8D).Remarkably, glucose induced significantly higher values of spermhyperactivation after starving. This experiment suggests that uponstarving, sperm can move better than when they are incubated with energynutrients the whole time.

In vitro fertilization and embryo development is enhanced using Starvingand Rescue method in young and old mice.

The significant increase in hyperactivated motility observed afterrescuing sperm incubated previously in starving media (starving plusrescue) suggested that this treatment can improve fertilization rates.To evaluate this hypothesis, the fertilizing capacity of sperm incubatedin control TYH capacitation media (CONTROL (C)) with sperm incubated inTYH media devoid of glucose and pyruvate and then rescued with theaddition of glucose and pyruvate (STARVING+RESCUE (S+R)) were compared.In mice, like in humans, decreased fertility has been observed in agedindividuals, and it is caused by different factors such as diet,exercise and genetic outcomes. A recent study has shown the effects ofadvanced paternal age on mice reproduction. Interestingly, the authorsconcluded that fertilization capacity (natural conception) is reducedwhen mice reach 12 months of age and it declines after that age.Remarkably, they also discover that IVF, in vitro embryo development,and embryo quality were also affected with age. Taking this intoconsideration, our experiments were conducted using male mice of threedifferent age groups: 3-6 month old, 6 to 12 month old, and 12-24 monthsold. For oocyte donors, young females were used (2 months old).

Initially these experiments were done using CD-1 oocytes (FIGS. 9 A, Band C) and then repeated with oocytes obtained from C57BL6 mouse strain(FIGS. 9D, E and F). The rationale of trying both types of oocytes isthat C57BL6 oocytes are not as good in fertilization protocols as theCD-1 oocytes. It was observed that the percentage of fertilized oocytes(measured by the percentage of oocytes arriving to 2-cell embryos) wasincreased when sperm are treated in S+R conditions (FIG. 9 A). Thehigher fertilization rates were maintained regardless the age of themice (3-6; 6-12, and 12-24 months old). Two-cell embryos obtained ineach condition were then transferred to KSOM media for further embryodevelopment. Surprisingly, the percentage of blastocysts achieved fromtwo-cell embryos obtained using S+R sperm was higher than the onesfertilized with C sperm (FIG. 9B). Notice that in FIG. 9B, thepercentage of blastocyst development is calculated from the total amountof two-cell embryos in each condition. If the percentage of blastocystdevelopment was plotted considering the initial number of oocytes, it ispossible to observe that the S+R treatment is highly efficient whencompared with standard media (FIG. 9C). Improvement in the percentage offertilization and embryo development was also observed when C57BL6oocytes were used (FIGS. 9D, E and F).

Experiments were then conducted to evaluate the speed by which spermtreated in standard conditions or starved and then rescue can fertilizeCD1 oocytes. Table 1A, first column, shows the incubation time of spermwith eggs for each treatment (control or starving+rescue) (secondcolumn). After this time period, eggs were removed, washed and continuethe incubation in fertilization media. The number of two-cell embryoswas evaluated the following day. The data presented in each of thecolumns indicate the number of oocytes (Oocytes #), the number ofoocytes reaching two-cell embryo stage (cleavage) with the respectivepercentage between parenthesis, the number of two-cell embryos reachingblastocyst stage (Blastocyst) with the respective percentage comparewith the initial number of two-cell embryos, and, finally, thepercentage of blastocysts taken into consideration the initial number ofoocytes in the assay (Total % blastocyst). In this table, it is possibleto observe that the starving plus rescue conditions increased thevelocity of fertilization.

Table 1 A and B. Comparison of standard and Starving plus Rescue spermtreatment regarding the speed of fertilization and the minimum amount ofsperm needed for insemination in vitro.

TABLE 1A Time Cleavage Blastocyst Total % (Min) Treatment Oocytes # (%)(%) Blastocyst 15 Control 298 23 (8) 15 (65) 5 30 278  91 (33) 64 (70)23 60 315 176 (56) 89 (51) 28 240 134 106 (65) 61 (61) 36 15 Rescue 285111 (39) 92 (83) 32 30 307 181 (59) 159 (88)  52 60 298 250 (84) 225(90)  76 240 112  98 (87) 87 (88) 78

TABLE 1B # Sperm Cleavage Blastocyst Total % Cells Treatment Oocytes #(%) (%) Blastocyst 500 Control 235 54 (23) 29 (54) 12 1000 260 80 (31)45 (56) 17 10000 245 88 (36) 33 (38) 13 100000 98 58 (60) 39 (67) 40 500Rescue 281 142 (47)  96 (73) 34 1000 220 145 (66)  107 (74)  49 10000255 181 (71)  152 (84)  60 100000 103 92 (90) 82 (89) 80

A similar experiment was conducted to evaluate the minimum number ofsperm needed for fertilization. This experiment is presented in Table1B. For this experiment, sperm incubated in control (Control) or instarving plus rescue (Rescue) media were counted and then a series ofdilution of the original sperm suspension were done with the purpose ofadding different number of sperm to the insemination drop as detailed incolumn 1. The columns present the same information described for Table1A: number of oocytes, number of two-cell (percentage), number ofblastocysts (percentage with respect to the number of two cells), andtotal % blastocyst with respect to the initial number of eggs. Data inTable 1B indicates that fertilization can be achieved with lower spermnumber.

Since the first successful IVF in mammals, it has become clear thatthere is a direct relationship between embryo quality and gestationalsuccess post embryo transfer. Different studies have shown that IVF havean impact on embryo development in vitro. Considering that the starvingmethodology improved fertilization rates and also increased the numberof blastocysts, it was decided to compare quality of blastocystsobtained with the different methodologies by counting the number ofcells present in 3.5 blastocysts. For each condition, 35 blastocystsobtained from 10 individual experiments were evaluated. The total numberof cells was assessed by counterstaining of nuclei and served as anindicator for division rates. While control sperm produced blastocystswith an average of 46 cells, starved and rescued sperm producedblastocysts with an average of 55 cells (FIG. 10 A). Blastocysts werefurther assessed by three-day outgrowth (OG) assay to test hatching,attachment and formation of the inner cell mass (ICM) with surroundingtrophoblast cells in an in vitro system. An n=10 outgrowth assays weredone from different 10 IVF experiments, and a total of 200 blastocystwere recorded for each of the sperm treatments. Control blastocystoutgrowth showed a significantly lower embryo attachment to the petridish (55%) than the Starving plus rescue treatment (75%) (FIG. 10B). Thenext step to evaluate embryo quality from 3.5 blastocyst embryosobtained with different sperm was to perform embryo transfer topseudo-pregnant mice females. 25 independent experiments were evaluated.For each experiment, the same number of 3.5 day blastocysts obtainedwith sperm treated either with control media or with starving plusrescue protocol were transferred non-surgically. Because, moreblastocysts were obtained routinely using the starving plus rescueprotocol, for these experiments, the number of blastocysts transferredwas always limited by the amount of blastocysts obtained withcontrol-treated sperm which range between 8 and 16 blastocysts. Aftertransfer, the females become pregnant and the litter size for eachcondition was recorded (FIG. 10C). Results were analyzed depending onthe mice age (2-3 months old vs 12-24 months old). As shown in FIG. 10C,the average litter size for embryos obtained using Starving plus Rescuesperm treatment was significantly higher for both age groups. The datawere also analyzed as percentage of pups per number of blastocyststransferred (FIG. 10D). Altogether these data indicate that blastocystsobtained using starved plus rescued sperm have better quality than thoseobtained using standard control conditions.

Intrauterine insemination (IUI) is an assisted reproductive techniquethat delivers sperm into the female tract bypassing the cervix. IUI isimplemented in humans as a fertility treatment, and widely performed incommercial breeding of livestock. However, in the literature there isfew studies about IUI in mouse compared with other species such ashumans, bovine, horses and many others. Therefore, it was decided toevaluate the success of sperm using control media vs the success ofsperm treated with the starving protocol. In IUI protocol, the rescue ofsperm was not done; the sperm were used directly after incubation inmedia without nutrients (starving). Because IUI in mice has been shownto work better when sperm are incubated in non-capacitating conditions,the media used for these experiments for both control and starvingconditions was done without addition of HCO3− and BSA. Success of IUI inmice depends on two main variables. The first one is the number offemales that becomes pregnant with respect to the total of femalesinseminated (FIG. 11 A). The second one is the litter size in thosefemales that become pregnant (FIG. 11B). As an example pups obtained inone of the IUI experiments using starved plus rescue protocol are shown(FIG. 11C).

As mentioned above, in-breed genetic backgrounds have lower fertility invivo and in vitro. These mice strains such as C57BL6 are relevant forresearch and high priority on the mouse phenome database. In additiongenetic manipulation of these valuable mice leads in many cases to acutesub-fertility. It was decided to use two knock out (KO) models(FerTDR/DR and Akita) and one in-breed mouse (SJL/J) with provensub-fertility in vitro. As it was shown previously calcium ionophoreA23187 overcome mice infertility in different KO models; therefore, forFerT KO, A23187 treatment was also used to compare the success rate ofdifferent treatments. FerT sperm fertilization rate was improved withA23187 transient incubation. However, starving treatment improvesfertilization of FerTDR/DR sperm to higher levels (FIG. 12 A). In bothtreatments, two-cell embryos were transferred to KSOM media and furtherincubated for 3.5 days. Results indicate that once two-cell areobtained, both ionophore treatment and starving plus rescue protocolsare equally successful for embryo development (FIG. 12B). Similarly,starving plus rescue improved fertilization rates of sperm from Akitaand SJL/J mice (FIG. 12C). In one of the experiments using FerTDR/DR andone using Akita mice, blastocysts obtained using starved plus rescueprotocol were transferred and pups were obtained (10 for FerTDR/DR and 5for Akita) (data not shown).

Many genes have been shown to play a role in fertilization. A group ofgenes code for the sperm-specific calcium channel complex CatSper.Knock-out mice lacking any of the four main CatSper subunits (CatSper1,CatSper2, CatSper3 or CatSper4) are infertile in vivo and in vitro. Ashort treatment with calcium ionophore A23187 induces fertilizationcapacity in sperm from these mice. Using this technique combined withembryo development and embryo transfer pups were obtained (Navarrete etal., Sci. Rep. 2016). To investigate if the starving plus rescuetechnology was also able to rescue the infertility phenotype, sperm wereincubated in four different conditions: 1) Control; 2) A23187 transienttreatment; 3) starved plus rescue treatment; and 4) starved plus rescuetreatment followed by A23187 transient treatment. After thesetreatments, sperm were combined with eggs and the number of two-cellembryos developed counted (FIG. 13 A). The use of A23187 rescued theinfertile phenotype. On the other hand, starved plus rescued spermincubation protocol did not rescue infertility. However, when bothtreatments were combined the CatSper fertilization rate was close to90%. In each of these experiments, two-cell embryos were transferred toKSOM media and further incubated for 3.5 days. Blastocyst were thencounted and plotted as percentage of blastocyst in relation to thenumber of two-cell embryos (FIG. 6B). In this case, the A23187 transienttreatment achieved 20% development and the combined starved plus rescuewith A23187 transient treatment was close to 90%. From theseexperiments, a total of 15 blastocysts from each treatment weretransferred into 2.5 days pseudo-pregnant females. A23187 alone gavebirth to three heterozygous pups and the starved plus rescue protocolgave birth to six heterozygous pups. Altogether, these results suggestthat combination of treatments is very effective in producing healthyembryos and that it can be used as treatment in some infertility cases.

Example V—Bovine Sperm

Media requirements for sperm in ART approaches vary greatly amongspecies and have been developed for each sperm type essentially by trialand error. However, in all cases sperm need to efficiently synthesizeATP. To investigate the extent by which metabolic regulation can be usedin other animal models, bovine sperm was used to conduct IVF, ICSI andembryo development experiments. These experiments were obtained usingfrozen sperm and in vitro matured embryo. IVF in bovines iswell-established and the efficiency of the method is around 60%. Controland metabolically modified sperm performed similarly in IVF (FIG. 14 A);however, an increased percentage of blastocysts were obtained with themetabolically treated sperm (FIG. 14B). Blastocysts from one of theexperiments are shown (FIGS. 14C and 14D).

Although as said, IVF methods are well-established in bovine,intracellular sperm injection (ICSI) is not effective using bovinesperm. This is due to deficiencies in the ability of sperm to induceCa2+ oscillations. Therefore, the only method available to do ICSI withbovine sperm is by treating embryos with pharmacological reagents suchas Ca2+ ionophores which activates the egg. Notice that one maindifference on the Ca2+ ionophore treatment herein is that in the instantcase, only the sperm are transiently exposed to this pharmacologicalreagent and that the reagent is washed out before the sperm become incontact with the egg. In an unexpected result, bovine sperm incubated instarved media and then exposed to nutrients acquire the ability toinduce calcium oscillations in bovine eggs after injection by ICSI (FIG.15 A) in significantly more number of injected eggs than controls (FIG.15B). Moreover, this method also increased significantly the percentageof two cell embryos (FIG. 16 A) and 2 blastocysts were obtained usingthis treatment (FIG. 16B). Although in comparison with mouse sperm datathe percentage of blastocysts appear low, it is important to highlightthat this is the first time that blastocyst are obtained using ICSIwithout exposing the embryo to pharmacological reagents.

The methods described herein find use in IVF, ICSI and artificialinsemination in humans and other mammals animals. However, each specieshas specific difficulties. For example, in humans, artificialinsemination is not used frequently despite being the less invasive andcostly method because the success rate is limited. The methods describedherein improve the outcome of intrauterine insemination as well as forvaginal insemination.

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The invention is described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin its scope. All referenced publications, patents and patentdocuments are intended to be incorporated by reference, as thoughindividually incorporated by reference.

What is claimed is:
 1. A method to increase Artificial Inseminationpregnancy rates comprising a) isolating biological fluid comprisingsperm; b) removing energy nutrients from said biological fluid in a); c)placing said sperm of a) and/or b) in a media absent energy nutrientsincluding glucose and pyruvate for a period of time sufficient for thesperm to lose progressive motility; d) adding an energy nutrient to saidmedia and sperm of c), wherein said energy nutrient is a glycolyticsubstrate or a Krebs cycle substrate or a combination thereof; and e)using said sperm from c) or d) for intrauterine or vaginal inseminationso as to increase Artificial Insemination pregnancy rates as compared toa method where sperm cells have not under gone energy nutrientstarvation.
 2. The method of claim 1, wherein the energy nutrient addedto said media in step d) is glucose, fructose, pyruvate, lactate,citrate or a combination thereof.
 3. The method of claim 1, wherein thesperm is from a vertebrate.
 4. The method of claim 1, wherein the spermcells are further exposed to an ionophore.
 5. The method of claim 1,wherein the ionophore is a calcium.
 6. The method of claim 1, wherein instep c) glycolytic and Krebs cycle metabolites are not present.
 7. Themethod of claim 1, wherein in step c) glucose, pyruvate and lactate arenot present.